JP4815602B2 - Dioxins and other responsive plasmids, dioxin and other transgenic cells, and detection methods and biosensors using the same - Google Patents

Description

  In the present invention, endocrine disrupting substances such as dioxins and polycyclic aromatic hydrocarbons and some carcinogenic substances act via an aromatic hydrocarbon receptor (hereinafter referred to as AhR) that is an intracellular receptor. The present invention relates to a simple, rapid and sensitive detection or quantification method for harmful chemical substances based on the above, a detection substance, and a dioxin detection biosensor.

  In order to establish countermeasures against environmental pollution, one of the serious problems of modern society, it is necessary to accurately evaluate the degree of exposure to harmful chemical substances in the environment. For this purpose, it is indispensable to establish a method for analyzing harmful chemical substances that is simple and has high sensitivity and reproducibility.

  Dioxins cause various harmful effects in a very small amount. Therefore, the establishment of a method for detecting dioxins sensitively and quickly is considered an urgent issue in order to accurately evaluate exposure to dioxins and prevent health problems. In addition, overall and quantitative evaluation of the biological toxicity of polycyclic aromatic hydrocarbons, which are harmful substances in the environment like dioxins, and tobacco smoke containing these in a complex manner is also a major issue.

  Typical methods for detecting dioxins in the environment include the use of endogenous biomarkers such as cytochrome P-4501A1, a drug metabolizing enzyme, bioassay using cultured cells, enzyme immunoassay, chromatography There are methods that use graphy. In particular, bioassays using genetic engineering techniques have recently attracted attention due to their simplicity and high sensitivity.

  Such genetic engineering bioassays are composed of several basic units. Such basic units are (1) cells and (2) gene structures to be incorporated into cells. Here, such a gene structure further comprises a dioxin-responsive DNA sequence, which is a DNA sequence that functions as a sensor for dioxins, and a marker protein gene that defines a marker protein.

  By introducing such a gene structure into a cell and stably incorporating it into the chromosomal DNA of the cell, it is possible to produce a sensor cell that reacts with dioxins and the like.

  When such transgenic cells are exposed to samples containing dioxins, dioxins, etc. first bind to intracellular AhR, and then form a complex with a coactivator (hereinafter referred to as Arnt), a transcription-promoting coupling factor. The complex activates a dioxin-responsive DNA sequence as a transcription factor. As a result, the expression of the marker protein gene downstream of the gene sequence is promoted.

  By expressing such a marker protein gene, a marker protein is produced, and by quantifying the protein, it is possible to evaluate how much dioxins or the like are present in the sample.

  As marker protein genes, enzyme genes such as chloramphenicol / acylribosyl transferase, β-galactosidase, luciferase, and green fluorescent protein genes have been used so far.

However, in conventional bioassays, (1) since marker protein is not secreted, it is necessary to destroy the cells and extract the marker protein for quantification. (2) Trace amounts of harmful substances in the environment Sensitivity is inadequate for detection, and even in high-sensitivity systems, 1, 3, 7, and 8-tetrachlorodibenzo-p-dioxin (TCDD) conversion, 1 pM is the limit of detection, and (3) 2 for bioassay 3-4 days required (4) 60,000 to 100,000 cells per sample are required, which can lead to high costs such as culture and labor costs. it can. In particular, (2) to (4) are serious obstacles to efficiently screening a large number of samples.
PA Behnisch, K. Hosoe, S. Sakai, Bioanalytical screening methods for dioxins and dioxin-like compounds: a review of bioassay / biomarker technology, Environ. Int. 27 (2001) 413-439. Japanese Patent Application No. 2004-135662 “Transformant for measuring dioxins and detection method, quantitative analysis method and screening method of dioxins using the same”

  Therefore, the present invention is compared with the conventional bioassay for detecting dioxins using luciferase or green fluorescent protein, (1) simplification of bioassay process, (2) improvement of detection sensitivity, (3) bioassay time. (4) Reduction of expenses such as culture costs and labor costs.

  The present invention incorporates a secretory marker protein gene downstream of a dioxin and / or polycyclic aromatic hydrocarbon hypersensitive gene sequence activated in response to dioxins and / or polycyclic aromatic hydrocarbons. It is a dioxin and / or polycyclic aromatic hydrocarbon responsive plasmid.

  The secretory marker protein gene is preferably a secretory alkaline phosphatase gene.

  In the present invention, the dioxins and / or polycyclic aromatic hydrocarbon-responsive plasmids described above are introduced into cultured cells that highly express an aromatic hydrocarbon receptor, and the aromatic hydrocarbon receptor is a dioxin and / or Alternatively, it is a gene-introduced cell for measuring dioxins and / or polycyclic aromatic hydrocarbons that binds to a polycyclic aromatic hydrocarbon and produces a secreted marker protein.

  The cultured cell that highly expresses the aromatic hydrocarbon receptor is preferably Hepa-1c1c7.

  The present invention provides a biosensor using the above-described gene-transferred cell for measuring dioxins and / or polycyclic aromatic hydrocarbons as a detection sensor for dioxins and / or polycyclic aromatic hydrocarbons.

  The present invention relates to a dioxins and / or polycyclic aromatic hydrocarbon highly sensitive gene sequence activated in response to dioxins and / or polycyclic aromatic hydrocarbons, and a secretory marker downstream of the gene sequences. Genes for measuring dioxins and / or polycyclic aromatic hydrocarbons produced by introducing dioxins and / or polycyclic aromatic hydrocarbon-responsive plasmids incorporating protein genes into cells that highly express aromatic hydrocarbon receptors The introduced cells are exposed to dioxins and / or polycyclic aromatic hydrocarbons, the activity of the secreted marker protein secreted by the transgenic cells is quantified, and the dioxins and / or polycyclic aromatic hydrocarbons are quantified. This is a method for detecting dioxins to be detected and / or polycyclic aromatic hydrocarbons.

  It is preferable that the secretory marker protein is secretory alkaline phosphatase. The cell is preferably Hepa-1c1c7.

  The present invention relates to dioxins and / or polycyclic aromatic hydrocarbons highly sensitive gene sequences activated in response to dioxins and / or polycyclic aromatic hydrocarbons, and a secreted type downstream of the gene sequences. For measuring dioxins and / or polycyclic aromatic hydrocarbons produced by introducing dioxins and / or polycyclic aromatic hydrocarbon-responsive plasmids incorporating a marker protein gene into cells that highly express aromatic hydrocarbon receptors To evaluate the biological toxicity of tobacco smoke by exposing the transgenic cell to tobacco smoke and quantifying the activity of the secreted marker protein secreted by the transgenic cell. Is the method.

  The secretory marker protein is preferably secretory alkaline phosphatase.

  According to the present invention, dioxins and polycyclic aromatic hydrocarbons can be easily measured by simple chemiluminescence measurement or the like. In addition, the dioxin measurement process from cell seeding to measurement can be completed in a few hours, and according to the present invention, low-cost and highly sensitive dioxins and / or polycyclic aromatic hydrocarbons can be measured. it can.

  The present invention is an excellent invention capable of detecting dioxins and / or polycyclic aromatic hydrocarbons contained in the atmosphere, rivers, soil, foods, and household equipment.

  The best mode for carrying out the invention will be described below.

  FIG. 1 shows a structural diagram of dioxins and / or polycyclic aromatic hydrocarbon reactive plasmid pDRE-SEAP10. pDRE-SEAP10 is a dioxin-sensitive DNA sequence (hereinafter MMTV-DRE11) that incorporates a dioxin-responsive DNA sequence (hereinafter DRE) into a part of the mouse breast cancer virus promoter (hereinafter MM TV) and downstream of it. It is composed of a secretory alkaline phosphatase (hereinafter referred to as SEAP) gene 12 and a polyadenylation signal (hereinafter referred to as poly A13) derived from simian virus 40 (hereinafter referred to as SV40).

  Conventionally, DRE has been used as a dioxin-responsive DNA sequence. In this example, MMTV-DRE in which DRE was incorporated into a part of MMTV was used as a sensor DNA sequence for sensing dioxins. This established a highly sensitive assay system.

  MMTV-DRE11 incorporates four DREs, which are known to be activated in response to dioxins, as part of the MMTV promoter. Responsive to dioxins that are stronger than those of DRE alone.

  Fig. 2 shows the results of a comparison of the sensor capabilities of MMTV-DRE and DRE. SEAP reporter plasmids having only MMTV-DRE and DRE as sensor sequences were prepared, and these genes were introduced into Hepa-1c1c7 cells. Details of gene transfer will be described later. These cells were then stimulated with TCDD and compared for increased SEAP activity. In cells into which a SEAP reporter plasmid having only DRE as a sensor sequence was introduced, SEAP activity increased 4.5-fold by TCDD stimulation.

  In contrast, SEAP activity increased 43.2 times in the cells into which the SEAP reporter plasmid having MMTV-DRE as a sensor sequence was introduced (Hepa1c1c7-DRE-SEAP cells, hereinafter referred to as HeDS cells) by TCDD stimulation. In cells into which a SEAP reporter plasmid having only MMTV as a sensor sequence was introduced, SEAP activity was not increased by TCDD. This means that the MMTV sequence strongly amplifies DRE response to dioxins. The high reactivity of MMTV-DRE enables rapid assay and low cost.

  In Example 1, SEAP was used as a marker protein. A bioassay system for dioxins using secreted marker protein has not been established so far, including SEAP. SEAP is a marker protein that can detect dioxins with high sensitivity along with luciferase, but unlike luciferase, SEAP is secreted outside the cell, so it is not necessary to destroy the cell and extract the protein.

  FIG. 3 is a graph in which the detection sensitivity of TCDD was measured with 5000 HeDS cells. The conventional dioxin assay using luciferase requires 60,000 to 100,000 cells to detect 1 pM TCDD, whereas 0.5 pM TCDD in 5000 HeDS cells, as shown in FIG. Can be detected.

  By using SEAP as a marker protein, dioxins can be assayed with only a very small sample of the culture supernatant of only 5 μl. The transcription level of SEAP gene 12 and the secretion level of SEAP protein correlate very well, and the activity can be easily detected and quantified by a chemiluminescence detection system such as a luminometer. PolyA13 is an essential gene sequence in the process of messenger RNA being made and then translated into protein.

  pDRE-SEAP10 is prepared by conventional genetic engineering methods. That is, the MMTV-DRE11 fragment excised and purified by a restriction enzyme is incorporated upstream of the SEAP gene 12 of the SEAP plasmid without a promoter using T4 DNA ligase.

  T4 DNA ligase is an enzyme that links the 5 ′ end and 3 ′ end of adjacent DNA strands. The prepared pDRE-SEAP10 is introduced into Escherichia coli to increase in quantity, and pDRE-SEAP10 is purified by a genetic engineering routine.

  The prepared pDRE-SEAP is introduced into Hepa-1c1c7 cells, which are mouse liver cancer cell lines, according to the procedure shown in FIG. 4 to establish stable recombinant cells. Here, the inventor selected Hepa-1c1c7 cells because (1) in order to establish a bioassay system for dioxins, cells that produce an aromatic hydrocarbon receptor (hereinafter, AhR) are used. This is because it is indispensable and (2) in order to establish a highly sensitive assay system, it is essential to select cells that highly express AhR.

  As a result of intensive studies from this viewpoint, the inventor has selected Hepa-1c1c7, a cell line derived from liver cancer cells. The procedure for introducing the prepared pDRE-SEAP into Hepa-1c1c7 cells and establishing stable recombinant cells is as shown in FIG.

After thoroughly washing 4 × 10 ⁶ Hepa-1c1c7 cells detached from the culture dish with trypsin with PBS, which is a phosphate buffered saline, a cuvette for electroporation (hereinafter, electroporation) No. 165-2088) (S1). Add 20 μg of pDRE-SEAP and 2 μg of plasmid pcDNA3.1 containing neomycin resistance gene, mix, and leave on ice for 10 minutes (S2).

Next, electroporation was performed under conditions of 150 mV and 960 μF using Gene Pulser (manufactured by Bio-Rad), and 1/10 to 1/20 of the number of cells was seeded in a 100 mm culture dish at 37 ° C., CO ₂ The cells are cultured for 3 days in the presence of 5% and 10% fetal bovine serum (hereinafter FBS) (S3).

  As a culture solution, α-MEM (manufactured by Gibco, product number 12561-056) was used. Subsequently, by culturing for 1 to 2 weeks in the presence of 500 μg / ml neomycin, non-recombinant cells die (S4), and only recombinant cells in which pDRE-SEAP is stably integrated into the chromosomal DNA survive and proliferate To form agglomerates.

  The cell clumps are individually detached by trypsin treatment and detached, and each of them is transferred to a 96-well culture plate with 2 holes, and the culture is continued for 1 week (S5). After the cells are saturated, the culture medium in each well is replaced with 100 μl of α-MEM containing 1% FBS, and cultured for 24 hours in the presence or absence of 10 pM TCDD. A predetermined SEAP assay is used to establish HeDS cells that react most sensitively to dioxins at low concentrations and are used in bioassays for detecting dioxins (S6). In this specification, this bioassay method is referred to as DRE-based Sensing of dioxin via Secreted Alkalined phosphatase (hereinafter, DRESSA method).

  FIG. 5 shows the response mechanism of established HeDS cells to dioxins. In HeDS cells, each component of pDRE-SEAP10, that is, MMTV-DRE11, and its downstream SEAP gene 12 and polyadenylation signal 13 are stably integrated in its chromosomal DNA. When these cells are exposed to dioxins 20, the dioxins 20 first cross the cell membrane and bind to AhR14 present in the cytoplasm.

  In addition, AhR14 forms a complex with Arnt15, a coactivator, enters the nucleus, binds to MMTV-DRE 11, and activates it. As a result, transcription of downstream SEAP gene 12 is promoted, messenger RNA16 is produced, translated into protein 18 by ribosome 17, and rapidly secreted extracellularly as SEAP protein19. Therefore, by measuring the activity of the SEAP protein in the culture supernatant, it is possible to quantify the level of dioxins exposed to the cells.

The detection procedure for dioxins in the sample is shown in FIG. 6 as a flowchart. First, HeDS cells are seeded at a density of 2 × 10 ⁴ / well in each well of a 96-well culture plate. As a culture solution, 100 μl of α-MEM containing 1% FBS is dispensed into each well (S1). After culturing for 24 hours to fix the cells to the bottom of each well, the culture medium is replaced, and 1 μl of a liquid sample containing TCDD is added (S2). After culturing for 24 hours, 5 μl of the supernatant is sampled (S3), and the SEAP protein activity is measured by the procedure described below (S4).

  The SEAP protein activity in the culture supernatant is quantified as follows. Add 15 μl buffer to 5 μl culture supernatant and incubate at 65 ° C. for 30 minutes to inactivate endogenous alkaline phosphatase activity. Add 20 μl of a buffer containing L-homoarginine, an inhibitor of endogenous alkaline phosphatase, leave it for 5 minutes, add 15 μl of substrate CSPD, leave it in the dark for 30 minutes, and then chemistry with a luminometer. Measure the luminous intensity.

  FIG. 7 shows HeDS cells stimulated with 6 pM TCDD, and the transition of SEAP protein activity in the culture medium thereafter was followed up to 10 hours over time. As a control, dimethyl sulfoxide (hereinafter, DMSO), which is a solvent of TCDD, was used. A significant increase in SEAP protein activity was observed 4 hours after the addition of TCDD, and gradually increased over time.

  FIG. 8 shows the subsequent transition of SEAP protein activity up to 72 hours. It can be seen that the increase in SEAP protein activity lasted up to 48 hours after stimulation and then reached equilibrium.

  FIG. 9 shows the results of examining the sensitivity of HeDS cells. HeDS cells were exposed to TCDD at a low concentration of 0 to 1 pM, and the increase in SEAP protein activity in the culture was examined. A significant increase in SEAP protein activity was already observed at 0.25 pM (250 fM).

  Thus, the DRESSA method can detect TCDD at a concentration of 1 pM or less, which is the detection limit of conventional bioassays. The inventors have confirmed that the SEAP protein activity in the culture medium increases in a concentration-dependent manner up to 100 pM.

  FIG. 10 shows the number of cells required for the DRESSA method. That is, the number of cells shown in the graph was seeded in a 96-well culture plate, the cells were exposed to 1 nM TCDD, and then the SEAP protein activity in the culture was measured. As shown in FIG. 10, it was revealed that 1nM TCDD can be sufficiently detected if 50 cells exist per sample (one hole). This corresponds to a number from 1/1000 to 1/2000 compared to the 60,000-100,000 cells / well required for conventional assays.

  Since the DRESSA method described above is performed simply, economically and in a short time, the detection procedure described above can be simplified as shown in FIG. That is, first, 50 μl of the culture solution containing 1 μl of sample is dispensed into each well of a 96-well culture plate, and immediately added HeDS cells suspended in 50 μl of the culture solution (total 100 μl) (S1).

  After 3 to 4 hours, the culture supernatant is sampled (S2), and the SEAP protein activity is measured (S3). By using this technique, 1 pM TCDD, which is the detection limit of the current method, can be detected within 4 to 5 hours. Hereinafter, this expedient method is referred to as a rapid DRESSA method. The rapid DRESSA method is comparable to the DRESSA method in terms of detection sensitivity.

  FIG. 12 shows the time course of the SEAP protein activity in the culture medium after HeDS cells were stimulated with TCDD (6 pM and 1 nM) by the rapid DRESSA method. In all cases, a significant increase in SEAP protein activity was already observed at 3 hours after addition of TCDD and gradually increased over time.

  FIG. 13 is a comparison of the sensitivity between the DRESSA method and the rapid DRESSA method. The same number of cells were exposed to 6 pM TCDD under fixed conditions in the DRESSA method and floating conditions in the rapid DRESSA method, and the subsequent increase in SEAP protein activity in the culture broth was compared over time. As a result, as shown in FIG. 13, there was no difference between the two in terms of the detection sensitivity of dioxins until 24 hours after stimulation.

  FIG. 14 shows the results after stimulation of HeDS cells with 1 μM 3-methylcholanthrene (3-MC), benzo [a] pyrene (B [a] P), and β-naphthoflavone (βNF), which are polycyclic aromatic hydrocarbons. , SEAP protein activity in the culture supernatant. In either case, a significant induction of SEAP protein activity was observed compared to DMSO, which was a control stimulus. In this way, HeDS cells also react with 3-MC, B [a] P, βNF), which are harmful chemical substances other than dioxins that act via AhR, and secrete SEAP protein.

  Tobacco smoke contains a combination of harmful chemical substances such as dioxins and polycyclic aromatic hydrocarbons. AhR combined with these toxic chemicals in tobacco smoke binds to DRE and exerts its biological toxicity. Therefore, it is possible to show the AhR activation ability of tobacco smoke as a value converted to the amount of TCDD causing the equivalent AhR activation. The converted value was defined as DRE-activating potential value (hereinafter referred to as DAP value), and the AhR activation ability of tobacco smoke was quantitatively evaluated as an indicator of the biological toxicity of tobacco smoke. This evaluation method is extremely useful for quantifying the degree of influence of smoking on health.

  Using 5 types of tobacco brands with tar content of 1,6,10,14,20 mg / tube, tobacco smoke is generated at a suction rate of 10.5 l / min. Dissolve in

  HeDS cells are seeded in a 96-well culture plate at 5000 cells / well, and the tobacco smoke extract (5 species, each n = 4) is added at a dilution of 100 to 1000 times. As a reference chemical substance, add 1 to 100 pM TCDD in the same manner. After 16 hours of culture, collect 5 μl of the culture supernatant and measure the SEAP activity.

A calibration curve showing the correlation between TCDD concentration and SEAP activity is created, and the concentration of harmful substances in each cigarette smoke extract is calculated based on this and converted to TCDD. Table 1 shows the total amount of AhR activation ability contained and shown as the DAP value.
As shown in Table 1, tobacco smoke showed a very high level of AhR activation ability. Although there is a nearly positive correlation between the tar content and the DAP value, when comparing the tar content of 1 mg and 20 mg, the former DAP value is not 1/20 that of the latter, Only about 1/3. On the other hand, the DAP value is higher on the 14 mg brand than on the 20 mg brand. These facts mean that tar content, which is generally used as an indicator of the degree of impact on health, is insufficient as an indicator of biological toxicity. The significance of the DAP value is high.

  The present invention is capable of detecting dioxins and similar harmful chemical substances easily, quickly, inexpensively and with super sensitivity. Industrial wastewater monitoring, drinking water quality control, food quality control, and monitoring of endocrine disrupting substances in industrial products. Industrial value is also high.

Structure of dioxins and other responsive plasmids (pDRE-SEAP) A comparison of the sensor capabilities of MMTV-DRE and DRE Figure of TCDD detection sensitivity measured with 5000 HeDS cells Procedure for HeDS cell establishment Dioxin sensing mechanism of HeDS cells Procedure for detection of dioxins by DRESSA method Transition of SEAP protein activity in culture supernatant after stimulation of HeDS cells with TCDD (short time) Transition of SEAP protein activity in culture supernatant after stimulation of HeDS cells with TCDD (long time) Graphical representation of elevated SEAP protein activity in culture medium after exposure of HeDS cells to TCDD at low concentrations of 0 to 1 pM Graphical representation of the number of HeDS cells required for the RESSA method Procedure for detection of dioxins by rapid DRESSA method Transition of SEAP protein activity in culture supernatant after cells were stimulated with TCDD by rapid DRESSA method Diagram showing comparison of detection sensitivity between DRESSA method and rapid DRESSA method Graphical representation of increased SEAP protein activity in culture supernatant after stimulation of HeDS cells with dioxin-like chemicals

Explanation of symbols

1 HeDS cells
10 Dioxin responsive plasmid (pDRE-SEAP)
11 Dioxin high sensitivity gene sequence (MMTV-DRE)
12 Secreted marker protein gene (SEAP)
13 Polyadenylation signal (polyA)
14 Aromatic hydrocarbon receptor (AhR)
15 Co-activator (Arnt)
16 Messenger RNA of SEAP gene
17 Ribosome
18 SEAP protein
19 Secretion of SEAP protein outside the cell
20 Dioxins

Claims (4)

A gene sequence (hereinafter referred to as DRE gene) in which a gene activated by reacting with dioxins and / or polycyclic aromatic hydrocarbons (hereinafter referred to as DRE gene) is incorporated into a part of a mouse mammary tumor virus promoter (hereinafter referred to as MMTV). (Hereinafter referred to as MMTV-DRE)) and a plasmid containing a secreted alkaline phosphatase gene downstream of the DRE gene into a mouse cancer cell line (Hepa-1c1c7) and / or dioxins and / or A transgenic cell for measuring polycyclic aromatic hydrocarbons.     A biosensor comprising the gene-introduced cell according to claim 1, wherein the gene-introduced cell is used for detection of dioxins and / or polycyclic aromatic hydrogen.     Dioxins and / or polycyclic aromatic hydrogens are detected by measuring the activity of a secreted marker protein secreted by the transgenic cells when the transgenic cells of claim 1 are exposed to an evaluation sample. A method for detecting dioxins and / or polycyclic aromatic hydrocarbons.     The biological toxicity of tobacco smoke is evaluated by measuring the activity of a secreted marker protein secreted by the transgenic cell when the transgenic cell according to claim 1 is exposed to tobacco smoke. To evaluate the biological toxicity of tobacco smoke.

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